Jorjani Biomedicine Journal

Highlights

• Administration of Linum Usitatissimum and aerobic exercise could exert dynamic cardioprotective properties through their antioxidant effects.
• Linum Usitatissimum and aerobic exercise significantly improve the 6-methyl guanine level in endothelial and heart cells.
• The interaction of Linum Usitatissimum and aerobic exercise significantly increased the ATP level in endothelial and heart cells.

Introduction
Cardiovascular disorders are one of the most prevalent life-threatening diseases on the globe. Based on reports, energy depletion and DNA damage in response to oxidative stresses play essential roles in the progression of cardiovascular, coronary diseases, ischemia-reperfusion, and heart failure disorders (1). These aberrations initiate or increase cellular death in cardiac tissues, leading to severe diseases and worsening the illness condition. In this regard, many studies have shown a high level of 6-methylguanine (a marker of DNA damage) and a low level of ATP in cardiac and related endothelial cells in patients with ischemia-reperfusion and heart failure illnesses (2). Moreover, the researchers have displayed attenuation of oxidative-induced death in cardiomyocytes, accompanies by a better prognosis in related patients (3).
Oxidative stresses could be formed in many physiologic and pathologic conditions, especially in high-energy demanding tissues such as the heart. The oxidative stresses producing reactive oxygen species (ROS) have been identified to be involved in many diseases, including cardiac disorders, as the oxidative stress markers are found elevated in cardiovascular diseases (4). Hydrogen peroxidase (H₂O₂) is one of the most prominent ROS engaged in the pathophysiology of cardiac illnesses. That is because of the high energetic demands of heart tissue supplied through oxidative phosphorylation (5). Hence, the heart tissue subsequently will be exposed to a high level of oxidative stresses so that ROSs are linked to different cardiac pathologic disorders, including hypertrophy and ischemia (6). The ROSs, especially H₂O₂, are the main factors leading to cellular apoptosis, necrosis, and tissue failure (7). The integrity of the genome is an essential factor for cell survival. The complexity of DNA replication and repair systems is responsible for maintaining this integrity (8). In addition to DNA damage caused by normal metabolism, further damages are caused by oxidative stresses. The damaged-DNA-induced cell death is just one of many biological consequences of oxidative stresses, including H₂O₂ (9). Studies show that H₂O₂ can impair nucleotide bases and biological responses immediately after DNA damage, including DNA repair and cell cycle arrest (10). More importantly, DNA damage activates caspases protein, a downstream target of the signaling network in DNA damage that gets stabilized and activated to stimulate the expression of pro-apoptotic genes (11, 12).
Moreover, recent evidence suggests that regular exercise has a considerable ameliorative role in cardiovascular diseases, notably its protective effects against oxidative stress-related damages (13, 14). Thus, this association has made a strong linkage between sedentary and various illnesses, especially cardiovascular diseases. Cardiovascular tissue is highly vulnerable to oxidative stress due to high oxygen consumption and insufficient antioxidant system (3,5). Hence, aerobic exercise has been identified to strengthen the antioxidative defense system, heart muscle, and blood volume, so that it has reported up regulating antioxidants enzymes (15).
Aerobic exercise (Ae) reduces mitochondrial oxidant production and increases mitochondrial antioxidant enzyme activity (13). de Meirelles et al reported (2014) reported that factors involved in cardiac cell survival pathways are significantly increased during aerobic exercise in the individual cells (14). Moreover, it has been indicated that aerobic exercise could reduce the risk of injury, oxidative stress, and inflammatory signaling in cardiac cells (15). Hence, exercise efficiently can decrease the production of ROSs such as H₂O₂ through improving antioxidant systems (16).
The Flaxseed or Linum Usitatissimum (Lu) belongs to the Linaceae family that grows widely in tropical and subtropical regions (17). It is one of the oldest cultivated crops in the world for its fiber, seeds, oil, and by-products (18). The flaxseed is also a rich source of vegetarian unsaturated omega-3 fatty acids (PUFA) and contains 50% alpha-linoleic acid, an omega-3 fatty acid. This plant is recognized as one of the most important medicinal herbs and has been scientifically paid attention to containing beneficial fatty acids, lignans, fiber, protein, and vitamins (19). These omega-3 fatty acids are vital for the proper function of the brain, nervous system, kidney, and sexual health (20). The most considerable lignan present in this plant, the Secoisolariciresinol diglucoside (SDG), also shows antioxidant, anti-apoptotic, anticoagulant, antiviral, antibacterial properties, and cytotoxic effects on some human cancerous cells, as well (21). Likewise, it has been indicated that Lu proteins effectively remove hydroxyl radicals, improving endothelial function due to their high arginine levels (22). Furthermore, regarding reports, Lu extract has been found to have high antioxidative traits because of contained vitamins and lignans (23-25).
Based on what is implied here, the current study aimed to investigate the simultaneous effect of aerobic exercise and flaxseed extract on apoptosis and DNA damage of endothelial aortic and heart tissue poisoned rats with H2O2. So that, the toxicity of hydrogen peroxide led to increased apoptosis as well as oxidative stress in the target cells. As oxidative stress could efficiently trigger DNA lesions and subsequently increase apoptosis in specific cells (26), we administered the Lu and aerobic exercise to rats to analyze their accompanying effects on attenuation of apoptosis in cells imposed to extensive oxidative stress. It is not far to efficiently use herbal medicine in therapeutical and preventive approaches in cardiovascular patients.
Materials and Methods

The ethics committee approved the whole experimental protocol in this study of Islamic Azad University, Mahallat Branch (IR.IAU.ARAK.REC.1399.043). It was done under the NIH (National Institutes of Health) guide for the care and use of laboratory animals (No. 80–23), which emphasized minimal animals being sacrificed and minimal pain imposed during the study.
The male Wistar Albino rats (10-12 weeks, 200 ± 20 g) were selected and purchased from Pasteur, Tehran, Iran. Based on various treatments, the rats were randomly and equally divided into 7 groups (n=8), including 1) Healthy Control (HC) who received no Lu extract and no Ae, 2) Lu1 (received 5 mg/kg of Lu supplementation), 3) Lu2 (received 10 mg/kg of Lu supplementation), 4) Ae (received only Ae), 5) Ae Lu1 (received 5 mg/kg of Lu supplementation as well as Ae), 6) Ae Lu2 (received 10 mg/kg of Lu supplementation as well as Ae), and 7) Toxicated Control (TC with no treatment). Afterward, they were separated into separate cages with optimized temperatures to 22-27 °C at the university’s Animal Care Facility. They were alternatively exposed to 12 hours of light and 12 hours of darkness during adaption and study periods with constant free access to water and food.

The peroxide hydrogen was used to poison the rats (27, 28), which was prepared from product oxidants 9% (Grape Oxidant 6 Number 1 Atusa 60 ml) purchased from Atusa Company, Tehran, Iran. The primary substance weighted 60 mg was transferred into a box with a volume of 125.123 mm² covered by a barrier net to prohibit oral usage by rats so that hydrogen peroxide was distributed throughout the cage air. The animals were entered into the cage at each turn so that rats inhaled the air inside the cage for 3 hours a day for one week.
 

The poisoned rats were treated with plant extract and aerobic exercise. The group that intoxicated control got intoxicated by H2O2. The Lu1 group received 5 mg/kg of herbal extract. The Lu2 group received 10 mg/kg of herbal extract. The Ae group underwent aerobic exercise. The Ae Lu1 group received 5 mg/kg of herbal extract as well as aerobic exercise. The Ae Lu2 group received 10 mg/kg of herbal extract as well as aerobic exercise. The HC group did not receive H₂O₂, herbal extract, and aerobic exercise. Although the extract was bitter, no sweetener was not used due to eliminating any probable chemical interactions. The extract was orally administered daily using a syringe to rats for 28 days (4 weeks), and groups Ae Lu1, Ae Lu2 received extract after every aerobic exercise session during weeks 2 and 3.

The low-intensity interval training (LIIT) was performed through 2 separate phases so that, primarily, the rats experienced an adaptation stage for 4 days, in which the rats ran 1 min at a speed of 20-25 m/min on a rotational bar 4 times a day. Phase 2 (the main phase) had consisted of 10 repeated programs. Each program consisted of 1 min running followed by 2 min active rest (running at half-speed). Hence, each session of the main exercise program was 30 minutes per rat. The main program lasted 4 weeks, 5 sessions weekly.

Primarily, 10 g of Linum Usitatissimum seeds (Pakan Bazr co, Isfahan, Iran) were thoroughly grinded. Next, the powder was mixed with petroleum ether solvent and put in a Soxhlet extractor for 10 hours for oil extraction. The sample lacking oil was wholly dried in the next step, and its methanolic extracts were purified via methanol using a Soxhlet extractor for 16 hours. The resulted yellow solution was as methanolic extract was kept at 50° C for 5 hours. After thorough evaporation of existed methanol, the yellow powder was remained and dissolved in normal saline. This extract is stored at 4 °C in darkness.

The tissues for molecular analysis were separated 24h after the last treating session, during which the rats have fasted for 14h. Then, the rats were passed out using ketamine (30-50 mg/kg) and xylazine (3-5 mg/kg) via intraperitoneal injection.

After assurance of anesthesia, a cleft was created at the breast of rats using a surgical blade through which the aortic endothelial and heart tissue were taken out and washed thoroughly by normal saline. Then, the tissues got immediately frozen by liquid nitrogen and stored at -80 °C. To homogenate collected tissue, after defrosting at RT, pieces of the left ventricle and beginning parts of the aortic artery were separated. The tissue was transferred into 2 ml microtubes, as 500 μl of lysis buffer (EPX-99999-000) is needed for 100 mg of tissue, through 615-705 μl of lysis buffer for 123-141 mg of tissue samples. Moreover, the lysis buffer contained antiprotease that prohibited protein denaturation. Then a 5 mm stainless steel bead was added to microtubes. Finally, the microtubes were transferred into the TissueLyser device (QIAGEN) by which Homogenization of sample was performed for 2 minutes at 25 Hz followed by centrifuge at 4 °C for 10 minutes. Subsequently, the supernatant was poured into new microtubes, and the sample was diluted at ration 10 mg protein/ml using 1X PBS. The product was kept at -80 °C. Eventually, we quantitatively assessed the level of 6-methylguanine (pg/ml) as a marker of DNA damage and ATP (µM/ml) in both cells via ELISA (Mabtech, Sweden).

The Kolmogorov-Smirnov test was used to determine the normality of the distribution. All results were expressed as mean±standard deviation. In order to analyze the data and investigate the inconsistencies of the observation amongst different groups, the two-way analysis of variance ANOVA method was used, which was then followed by the LSD post hoc test. Biochemical data were analyzed using the Prism 8 software at a statistically significant level
 (P ≤ 0.05).
Result

The intragroup analyzes showed that the Linum Usitatissimum supplementation and aerobic exercise significantly reduced the levels of
6-methylguanine (6MG) in aortic endothelial (F=111.3, P=0.0008, ƞ=0.9823) and heart tissue (F=147.9, P=0.0005, ƞ=0.9867). Though based on intergroup analyses, the groups Lu1, Lu2, Ae, and AeLu1 for aortic endothelial cells and groups Lu1, Lu2, and Ae, for heart tissue cells, have been demonstrated did not show any differences in the level of 6MG compare to TC. On the contrary, the group Ae Lu2 for aortic endothelial cells and Ae Lu1 and Ae Lu2 for heart tissue cells showed a significantly lower level of 6MG (Figure 1). Thus, the most considerable response was observed when both interventions were simultaneously administered, especially in a high dose of Linum Usitatissimum.

 
Table 1. The mean concentration of 6MG in different groups receiving Lu and Ae in aortic endothelial and heart tissue cells. 2-way ANOVA analyzed the data.

*Healthy Control (HC) who received no Lu extract and no Ae, Lu1 (received 5 mg/kg of Lu supplementation), Lu2 (received 10 mg/kg of Lu supplementation), Ae (received only Ae), Ae Lu1 (received 5 mg/kg of Lu supplementation as well as Ae), Ae Lu2 (received 10 mg/kg of Lu supplementation as well as Ae), Toxicated Control (TC with no treatment). F: Statistical parameter of analysis of variance. P: significant statistical level. Ƞ: Effect size.
 

Figure 1. The mean concentration of 6-methylguanine in different groups receiving Lu and Ae.

*Healthy Control (HC) who received no Lu extract and no Ae, Lu1 (received 5 mg/kg of Lu supplementation), Lu2 (received 10 mg/kg of Lu supplementation), Ae (received only Ae), Ae Lu1 (received 5 mg/kg of Lu supplementation as well as Ae), Ae Lu2 (received 10 mg/kg of Lu supplementation as well as Ae), Toxicated Control (TC with no treatment).

Moreover, the intragroup analysis presented that the Linum Usitatissimum extract and aerobic exercise also increased ATP levels in the studied groups in both aortic endothelial (F=342.6, P=0.0003, ƞ=0.9942) and heart tissue (F=135.1, P=0.0013, ƞ=0.9854). Although, the intergroup analyzes displayed that the level of ATP did not differ statistically significant in Lu1, Lu2, and Ae groups for both cell types, while a significantly higher level of ATP was found in the Ae Lu1 and Ae Lu2 groups for both tissue types (Figure 2).
 

Table 2. The mean concentration of ATP in different groups receiving Lu and Ae in aortic endothelial cells.

*Healthy Control (HC) who received no Lu extract and no Ae, Lu1 (received 5 mg/kg of Lu supplementation), Lu2 (received 10 mg/kg of Lu supplementation), Ae (received only Ae), Ae Lu1 (received 5 mg/kg of Lu supplementation as well as Ae), Ae Lu2 (received 10 mg/kg of Lu supplementation as well as Ae), Toxicated Control (TC with no treatment). F: Statistical parameter of analysis of variance. P: significant statistical level. Ƞ: Effect size.

Figure 2. The mean level of ATP is displayed in different groups.

*Healthy Control (HC) who received no Lu extract and no Ae, Lu1 (received 5 mg/kg of Lu supplementation), Lu2 (received 10 mg/kg of Lu supplementation), Ae (received only Ae), Ae Lu1 (received 5 mg/kg of Lu supplementation as well as Ae), Ae Lu2 (received 10 mg/kg of Lu supplementation as well as Ae), Toxicated Control (TC with no treatment).

Discussion
Due to beneficial properties such as a lack of side effects, therapeutic approaches refocus on herbal therapies to prevent or improve diseases. Some known herbs have undergone many studies by which their pharmacologic characteristics have been proved (29). The Linum Usitatissimum is one of these well-known plants with numerous activities, including anti-cancer, anti-inflammation, antioxidant, and anti-apoptosis features.
To date, researches have shown that oxidative stresses play a significant role in cardiovascular disease through directing the cardiac cells to death (30). Because oxidative stresses like H₂O₂ can seriously damage cellular macromolecules such as DNA and also trigger ATP depletion through changing mitochondria membrane permeability and speeding up the respiratory electron transport, these changes can impose the high-energy demanding cardiac cells to stress, which eventuate in the activation of apoptotic cascade and cellular death (31). The antioxidant-promoting effect of exercise has been shown in many studies (13). Aerobic exercise has been found to protects cells against oxidative stress through strengthening the antioxidant system, such as up regulation of antioxidant enzymes and free radical scavengers (14, 15). The advantages of exercise are mediated via adaptation phenomenon, including modifying alterations in cardiorespiratory and musculoskeletal systems and body metabolism, which is best in aerobic exercise (14, 16).
Moreover, the antioxidant properties of Linum Usitatissimum have been verified in healthy cells (23, 32). Likewise, it has been displayed that Linum Usitatissimum exerts an anti-apoptotic solid effect on the healthy cell. In this regard, one of the most important mechanisms that Linum Usitatissimum executes its productive cellular effects is suggested to be its antioxidative trait (33). As noted, contained chemicals in this plant, such as lignans, have potential antioxidant effects responsible for attenuating apoptosis in normal cells. As we know the cardiovascular disorders such as ischemia-reperfusion, cardiac hypertrophy, and heart failure are discerned by extensive death of cardiomyocyte cells in these patients (34).
The present results displayed that both aerobic exercise and Linum Usitatissimum extract declined the DNA damage marker,
6-methylguanine, and led to a higher level of ATP in cells. As observed, the most desirable response was seen when both interventions were administered concurrently. A high concentration of plant extract was significantly effective without any exercise. In contrast, we did not recognize any significant alteration when aerobic exercise was applied without herbal extract. Thus, it can be seen here that these interventions could enhance each other’s effects so that we may obtain the aimed therapeutical outcome with less concentration of herb accompanied with aerobic exercise. The current study demonstrated that the methanolic extract of Linum Usitatissimum could reduce the severity of DNA damage, so that it might be suggested that antioxidant and anti-inflammation features of compounds existing in herbal extract could lessen the free radicals and ROS s such as H₂O₂ that firmly damage the cell genome and deplete mitochondrial ATP. As the H₂O₂ was implemented to poison rats, studies show that hydrogen peroxide, as one most potential detrimental molecules harms DNA via oxidative molecules’ production and inhibits DNA repair systems (35, 36). These oxidants result in alterations of purine and pyrimidine bases, which cause a single-chain and double-chain breakdown of the DNA molecule (36). hence it could be reported that molecules such as lignans and Secoisolariciresinol Diglucoside (SGD) in Linum Usitatissimum may be able to do two main functions; 1) stimulate DNA repair systems (37) and 2) lower the ROSs molecules like H₂O₂ to prevents DNA damages and  ATP synthesis disruption (38). Parallel to our study, Liu et al. mentioned that lignans molecules could effectively abate oxidative-induced DNA damages in rat liver tissue (39). Harper et al. also showed that the lignans compounds could protect cell DNA against oxidative damage (40).
Furthermore, Ghule et al. also note that Lu extracts decreased ROS products and the conservation of antioxidant enzymes in the renal artery (41). Based on our data and other studies, it could be suggested that the most probable assumption for the cardioprotective feature of Linum Usitatissimum could be its potential scavenging ability which is followed by minor DNA damage. In accordance with our results, Adolph et al. noted that molecule SGD lowers the cardiovascular risk factors according to its antioxidant and anti-inflammatory activities (42).